The long-term goal of this project is to elucidate the pathways of alphavirus RNA genome repair and remodeling leading to the generation of atypical viruses in infected host cells. Alphaviruses are an important cause of emerging viral encephalitides in animals and humans and are significant biodefense agents. Sindbis virus (SIN) is a human alphavirus and is one of the best studied model system for RNA genome structure, function and evolution. We recently demonstrated the biogenesis of a library of atypical SIN genomes that carry non-canonical A/U-rich RNA motifs at the 3'nontranslated region of SIN genome (3'NTR). Despite the absence of a known classical polymerase recognition site, these atypical SIN genomes are strikingly stable and infectious for generations in both vertebrate and mosquito cells in culture and in newborn mice. Based on our published work, we hypothesize that, a) viral replicase has the intrinsic ability to remodel alphavirus genomes by reiterative copying of specific RNA motifs by polymerase slippage;and b) alphavirus genomes are composed of functionally redundant, but no identical RNA motifs that recruit template RNA for RNA synthesis. To test these hypotheses, we propose the following specific aims: 1. To study how nucleotide homology regulates polymerase slippage and remodeling of SIN 3'NTR with repetitive sequence elements (RSE) 2. To identify and characterize RNA elements that recruit template RNA to initiate RNA synthesis from atypical SIN genomes. 3. Determine the potential of viral replicase expressed from an atypical genome to recognize and replicate other atypical SIN genomes. These studies are new to alphaviruses, and are expected to provide a genetic basis for the biogenesis of alphavirus genomes and maintainance in nature. Delineation of these pathways of virus evolution should lead to development of strategies to control emergence of outbreaks of alphaviruses and perhaps other mosquito-transmitted RNA viruses. Since alphaviruses are vigorously pursued as gene therapeutic and vaccine delivery vehicles, these studies will also be useful in the development of improved RNA vectors.